Antiproliferative effects of mitochondria-targeted N-acetylcysteine and analogs in cancer cells

N-acetylcysteine (NAC) has been used as an antioxidant drug in tumor cells and preclinical mice tumor xenografts, and it improves adaptive immunotherapy in melanoma. NAC is not readily bioavailable and is used in high concentrations. The effects of NAC have been attributed to its antioxidant and redox signaling role in mitochondria. New thiol-containing molecules targeted to mitochondria are needed. Here, mitochondria-targeted NAC with a 10-carbon alkyl side chain attached to a triphenylphosphonium group (Mito10-NAC) that is functionally similar to NAC was synthesized and studied. Mito10-NAC has a free sulfhydryl group and is more hydrophobic than NAC. Mito10-NAC is nearly 2000-fold more effective than NAC in inhibiting several cancer cells, including pancreatic cancer cells. Methylation of NAC and Mito10-NAC also inhibited cancer cell proliferation. Mito10-NAC inhibits mitochondrial complex I-induced respiration and, in combination with monocarboxylate transporter 1 inhibitor, synergistically decreased pancreatic cancer cell proliferation. Results suggest that the antiproliferative effects of NAC and Mito10-NAC are unlikely to be related to their antioxidant mechanism (i.e., scavenging of reactive oxygen species) or to the sulfhydryl group-dependent redox modulatory effects.

) and complex I-induced oxygen consumption were assessed using the Seahorse technique 18,19 . MiaPaCa-2 cells were treated with varying concentrations of NAC and Mito 10 -NAC, and the overall oxygen consumption rate (OCR) was measured (Fig. 6A). The usual bioenergetic indices for mitochondrial stress were monitored 19 . As shown, Mito 10 -NAC inhibited 50% of the basal OCR at the 20 µM level, whereas NAC required IC 50 values at much higher concentrations (> 100 mM) to inhibit the basal OCR. Because treatment with NAC over a 24 h time period caused considerable cell death (Fig. 5B), we tested to see if NAC or Mito 10 -NAC could directly inhibit mitochondrial complex I-dependent OCR (Fig. 6B) by injecting NAC or Mito 10 -NAC into permeabilized cells in real time. Under these conditions, NAC inhibited only about 30% of complex I-induced oxygen consumption, even up to concentrations of 100 mM; direct cell toxicity is observed at such concentrations (Fig. 5B). In contrast, the IC 50 value at which Mito 10 -NAC directly inhibits complex I-induced oxygen consumption is 37 µM. At this concentration of Mito 10 -NAC, there was negligible cell toxicity (Fig. 5B). Thiol-based antioxidants (NAC and glutathione [GSH] esters) induced transient mitochondrial oxidation and inhibition of the mitochondrial respiratory complex III in several cancer cells including glioblastoma 25,26 . However, the present results using real-time monitoring of mitochondrial complex I-induced oxygen consumption indicate that NAC had no effect on mitochondrial respiration, even at high concentrations, in MiaPaCa-2 cells.   www.nature.com/scientificreports/ Previously, we showed that simultaneous inhibition of monocarboxylate transporter 1 (MCT-1) and mitochondrial OXPHOS synergistically inhibited the proliferation of several cancer cells 30 . More recently, these findings were confirmed in a B-cell lymphoma xenograft using AZD3965 and another OXPHOS inhibitor 31 . NAC was reported to inhibit monocarboxylate transporter 4 (MCT-4) expression in cancer cell lines 32 . NAC decreased MCT-4 stromal expression that is used as a biomarker of breast cancer 9,33 . We surmised that Mito 10 -NAC may synergize with NAC. We compared the synergistic effects of Mito 10 -NAC with AZD3965, an MCT-1 inhibitor that is undergoing a Phase I/II clinical trial for cancer therapy. MiaPaCa-2 cells were treated with Mito 10 -NAC and AZD3965 or NAC, independently and together, and cell growth was monitored continuously. These results, presented in Fig. 7, indicate that Mito 10 -NAC is synergistic with MCT-1 inhibitors (AZD3965) but not with NAC, as shown by the combination-index-fraction affected plots.

Discussion
Relative inhibitory effects of mitochondria-targeted drugs. We previously reported that increasing the aliphatic chain length in TPP + -conjugated molecules greatly enhanced the antiproliferative potencies in tumor cells 18 . As shown in Fig. 3, the fold difference between the parent compound and the TPP + -modified compound (with 10 carbons in the linker side chain) is dependent on the parent compound, especially its hydropho-  Figure 3 shows the dose response characteristics of NAC, metformin (Met), lonidamine (LND), honokiol (HNK), and atovaquone (ATO) and the TPP + -modified analogs in MiaPaCa-2 cells 18,34,35 . The difference in antiproliferative effect between ATO and mitochondria-targeted ATO (Mito-ATO) is 85-fold, whereas the difference between NAC and Mito 10 -NAC is 1600-fold and between Met and Mito 10 -Met is 3300-fold. Although many factors are responsible for the fold difference between the TPP + -modified drug and the unmodified drug, the hydrophobicity of the parent drug is a major factor. If the parent compound is very hydrophilic (NAC), TPP + modification will likely induce a greater fold difference in antiproliferative effect and inhibition of mitochon-   36 due to the more negative mitochondrial membrane potential of tumor cells as compared with normal cells 20,21,[37][38][39] . We have previously shown that TPP + incorporation to the mitochondriatargeted drug is essential for its mitochondrial accumulation and antiproliferative efficacy in cancer cells 35,36,40 . www.nature.com/scientificreports/ Lack of radical scavenging mechanism. The paradoxical effects of reactive oxygen species (e.g., superoxide and hydrogen peroxide) have previously been reported in cancer cells 12,[41][42][43] . Superoxide and hydrogen peroxide, at low levels, are reported to promote tumorigenesis and tumor progression, but at higher levels, these species induce cytotoxicity in tumor cells and inhibit metastasis 12,43,44 . This suggests that reactive oxygen species inhibition will affect tumorigenesis, tumor progression, and metastasis differently. Redox modulators (NAC) and chain-breaking antioxidant-inhibiting lipid peroxidation (vitamin E) enhanced metastasis of lung cancer in mice 45,46 . However, based on the results obtained with methylated Mito 10 -NAC, we conclude that the reactive oxygen species or redox modulating effects of Mito 10 -NAC are unlikely to play a key role in its antiproliferative mechanism. The IC 50 value for Mito-NAC-SMe (lacking the -SH group) to inhibit cell proliferation (Fig. 7) is similar or slightly lower than that of Mito-NAC (having the -SH group). Regardless of the presence or absence of the redox-sensitive -SH group, the antiproliferative effects of Mito-NAC and Mito-NAC-SMe are unaffected. We have shown in other studies that blunting the nitroxide moiety (i.e., removing the superoxide dismutase mimetic mechanism) in Mito-CP did not affect its antiproliferative effect 47 . Recently, we showed that reactive oxygen species generation is not responsible for the antiproliferative effects of TPP + -based mitochondria-targeted drugs in cancer cells 48 .

Immunomodulatory effects of NAC and anti-tumor immune function.
Recently, NAC has found new applications in immunotherapy 5,6 . Chimeric antigen receptor (CAR) T cells are genetically modified T cells that will recognize and destroy a protein on cancer cells. CAR T cell therapy involves reprogramming a patient's own T cells to recognize and attack a specific protein in cancer cells, and then infusing the T cells back into the patient 49,50 . Often, enhanced oxidant-induced modifications in CAR T cells decrease this ability. NAC has been shown to improve the efficacy of adoptive T cell immunotherapy to treat melanoma 5,6 . In a recent study, the NAC T cells were cultured before they were infused as immunotherapy in a preclinical model of melanoma; this resulted in an improved outcome 5 . T cells treated with NAC were 33-fold more effective than those cultured without NAC. NAC improves the anti-tumor function of exhausted T cells, thereby enhancing therapeutic outcomes for adoptive cell transfer (ACT) therapy 6 . NAC activates PI3K/Akt, inhibiting Foxo1, and inhibits reactive oxygen species, thereby enhancing the antitumor functionality of T cells 6 . The NAC-mediated opposing effect on T cells was dependent on the concentration. At low concentrations, NAC had an immunostimulatory effect, and at higher concentrations NAC had a suppressive effect 7 . A Phase I clinical trial of NAC, which aims to optimize the metabolic tumor microenvironment, is ongoing 8 . Although the bioavailability of NAC is low, it is membranepermeable and has been shown to cross the blood-brain barrier in humans and rodents 10,11 . Because Mito 10 -NAC is considerably more effective in tumor cells, it is of interest to explore the possibility of enhancing CAR T cell therapy using Mito 10

Synergistic antitumor effect of OXPHOS inhibitors and MCT-1/4 inhibitors.
The antiproliferative effect of Mito 10 -NAC was enhanced in the presence of AZD3965, an inhibitor of MCT-1 transporter. The extent of the combinatorial effect is consistent with our previously published heat map representation for other mitochondria-targeted drugs 30 . AZD3965 has been reported to enhance intracellular acidosis through increased intracellular lactate and decreased extracellular lactate 53 . Relatively higher concentrations of AZD3965 were used to inhibit cancer cells. At these concentrations, AZD3965 exerts deleterious side effects. The combination therapy with mitochondria-targeted drugs is a promising option as it could significantly lower the concentration of AZD3965 used in cancer therapy. Other mitochondria-targeted drugs (i.e., metformin and phenformin) have been used in combination with AZD3965 in brain cancer studies 54 . However, their effective concentrations were widely different. In this study, Mito 10 -NAC was used at low micromolar levels in combination with AZD3965 to achieve a synergistic inhibition in proliferation.
Other considerations. The hyperpolarized [1-13 C] NAC probe of the hyperpolarized 13 C-MRI showed global distribution of NAC, including to the brain 55 . This finding is consistent with previous studies that used isotopically substituted NAC 56 . Both tumor cell and mice xenograft studies showed that [ 13 C] NAC forms a [ 13 C] NAC-GSH dimer as well as other homodimers. Further, because no detectable levels of the levels of [ 13 C] GSH were found, there is a possibility that NAC could induce GSH synthesis by an indirect mechanism 55 . Additionally, it is suggested that a shortened relaxation time could be the reason no [ 13 C] GSH was detected 57 . Induction of GSH by NAC is cell dependent, and the mechanism by which GSH forms has yet to be determined.
The cysteine residue, Cys90, in the ND3 subunit of mitochondrial complex I is reported to play a key role in mitochondrial function 23 . Glutathionylation or nitrosation of this critical cysteine residue regulates redox signaling 58 . Mito 10 -NAC could inhibit complex I by thiolation of mitochondrial cysteine proteome. However, results obtained from using the methylated analog of Mito 10 -NAC indicate that the disruption of mitochondrial cysteine proteome is probably not responsible for Mito 10 -NAC-induced inhibition of mitochondrial respiration. The exact target of Mito 10 -NAC and other analogs in mitochondria still needs to be determined.
It is possible that Mito-NAC-mediated antiproliferative effects are due to cell cycle arrest. Previously, it was reported that mitochondria-targeted drugs (e.g., Mito-magnolol) decreased AKT and Foxo1 phosphorylation and induced cell cycle arrest in the G1 phase of the cell cycle 59  www.nature.com/scientificreports/ Mito-NAC, and their methylated analogs on AKT signaling and cell cycle arrest in cancer cells should enhance our understanding of these redox-sensitive thiols. This study has several limitations. All the experiments were performed in different cancer cell lines. Based on the previous publications in which mitochondrial OXPHOS inhibitors were translated to in vivo mouse xenograft models 18,19,36 , we believe that Mito 10 -NAC and analogs will show similar potency in mouse xenografts as well. Given the current interest in the immunomodulatory effects of NAC, future studies should focus on the effect of Mito 10 -NAC and other related analogs in activated immune cells and immune competent mice. Results indicate that Mito-NAC remained relatively stable over the time course in cell proliferation experiments. However, a comprehensive analytical study is required to monitor any oxidative degradation of compounds over the experimental duration.

Methods
The methods used in the synthesis, cell experiments, and statistical analysis of Mito 10 -NAC and its analogs follow standard scientific methods we routinely use in our labs, and have been described previously 18,19,22,30,48,60 . Application of these methods to Mito 10 -NAC and its analogs are further described in the subsequent sections.
A stirred solution of N-acetyl-S-trityl-l-cysteine (0.3 g, 0.74 mmoL) in CH 2 Cl 2 /N,N-dimethylformamide (DMF) (15 mL/100 mL) was cooled to 0 °C and successively treated with HOBt (0.2 g, 1.48 mmoL) and DIC (0.19 g, 1.50 mmoL). After stirring for 2 h at room temperature, (10-aminodecyl)-triphenylphosphonium bromide hydrochloride (0.35 g, 0.65 mmoL) and triethylamine (188 μL, 0.13 mmoL) were added to the mixture. The reaction mixture was stirred overnight at room temperature. Then, CH 2 Cl 2 was added to the mixture as well as water (H 2 O) (25 mL). The organic layer was dried over sodium sulfate (Na 2 SO 4 ). The solvent was removed under reduced pressure. The crude product was poured in 100 mL of ether and centrifuged. The insoluble salt was collected and purified by flash chromatography (CH 2 Cl 2 /ethanol [EtOH] 9/1) and led to the corresponding trityl-Mito 10 -NAC (0.41 g, 71% yield). High-performance liquid chromatography-mass spectrometry (HPLC-MS) indicated that the product was sufficiently pure and could be used without further purification. Electrospray ionization-mass spectrometry (ESI-MS) for trityl-Mito 10  NMR spectra for Mito 10 -NAC are shown in Fig. S2.

Synthesis of Mito 10 -NAC-SMe.
The mitochondria targeted N-acetyl methylated cysteine (Mito 10 -NAC-SMe) was prepared using the same reaction conditions as for the synthesis of Mito 10 -NAC. The synthesis of Mito 10 -NAC-SMe is shown in Fig. 9. A stirred solution of N-acetyl-S-methyl-l-cysteine (0.15 g, 0.75 mmoL) in CH 2 Cl 2 /DMF (15 mL/100 mL) was cooled to 0 °C and successively treated with HOBt (0.13 g, 0.96 mmoL), DIC (152 µL, 0.96 mmoL). After stirring for 2 h at room temperature, (10-aminodecyl)-triphenylphosphonium bromide hydrochloride (0.35 g, 0.65 mmoL) and triethylamine (188 µL, 0.13 mmoL) were added to the mixture. The reaction mixture was stirred overnight at room temperature. Then, CH 2 Cl 2 was added to the mixture as well as H 2 O (25 mL). The organic layer was dried over Na 2 SO 4 . The solvent was removed under reduced pressure. The crude product was poured in 100 mL of ether and centrifuged. The insoluble salt was collected and purified by reverse phase chromatography  NMR spectra for Mito 10 -NAC-SMe are shown in Fig. S2. A mixture of trityl-Mito-PEG-NAC (0.2 g, 0.2 mmoL), triethylsilane (a few drops), in trifluoroacetic acid (1 mL) and CH 2 Cl 2 (1 mL) was stirred at room temperature for 1 h. Then, the mixture was purified directly by reverse phase chromatography on a C18 column (H 2 O/CH 3 CN from 9/1 to 0/10 with 0.1% of TFA) delivered the corresponding Mito-PEG 4 -NAC (0.14 g, 96% yield).  Cell proliferation measurements. The IncyCyte Live-Cell Analysis System was used to monitor cell proliferation 22,60 . This imaging system is noninvasive and enables continuous monitoring of cell confluence over several days. In a 96-well plate, cells were plated at 1000 cells per well in triplicates and left to adhere overnight. Cells were then treated with compounds tested at indicated concentrations, and the cell confluency was recorded over several days in the IncuCyte Live-Cell Analysis System.

Intracellular ATP levels.
After seeding cells, overnight at 20,000 per well in 96-well plates, cells were exposed to NAC analogs for 24 h. A luciferase-based assay was used to measure intracellular ATP levels as per the manufacturer's instructions (Sigma Aldrich, St. Louis, MO, Cat# FLAA). Briefly, an ATP assay mix solution consisting of luciferase and luciferin (Cat# FLAAM) was added to cell lysates. After swirling, the amount of light produced was immediately recorded in a luminometer. The results were normalized to the total protein level measured in each well, as determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA).
Mitochondrial respiration measurements. Mitochondrial oxygen consumption was measured in the Seahorse XF-96 Extracelluar Flux Analyzer (Agilent, North Billerica, MA) 18,19,48 . The bioenergetic function assay was used to determine the intact cell mitochondrial function of cells in response to drug treatment 18,19,48 . After cells were treated with NAC or Mito 10 -NAC for 24 h, eight baseline OCR measurements were taken before injection of oligomycin (1 mg/mL) to inhibit ATP synthase, dinitrophenol (50 µM) to uncouple the mitochondria and yield maximal OCR, and inhibitors of complexes I and III (1 µM rotenone and antimycin) to inhibit mitochondrial respiration. From these measurements, mitochondrial function indices were determined 18,19,48 . For mitochondrial complex I activity measurements, the mitochondrial complex I-induced OCR measurements were carried out in permeabilized cells in the presence of complex I substrates pyruvate/malate and complex II inhibitor malonate (10 mM) 18,19,48 . The IC 50 values were determined as previously reported 19,30,48 . Statistical analysis. Comparisons between the control and treatment groups were made using an unpaired Student's t-test analysis. P values of less than 0.05 were considered to be statistically significant. All values provided represent mean ± standard deviation. The number of replicates per treatment group are shown as n. The IC 50 values and fitting curves were calculated using OriginPro 2016 (OriginLab Corporation, Northampton, MA).

Data availability
This study did not generate/analyze any computational datasets/code nor publicly archived datasets.